Summary Understanding the mechanisms of cell migration is a fundamental question in cell, developmental and cancer biology. Decades of research has shown that the molecular underpinnings of cell migration are complex and the physical mechanisms driving migration are diverse. We have shown that depending on the local microenvironment, cell migration can be driven by actin polymerization as well as an osmotic gradient-driven water flux external to the cell. This so-called osmotic engine model (OEM) is prominent when cells are in tightly confined spaces. In vivo, cells migrate within diverse microenvironments, ranging from dense 3D extracellular matrices to narrow microchannels present in tissue, to complex somatic spaces with various kinds of physical obstacles. An open and un-addressed question is what are the important variables that dictate the relative contribution of actin polymerization-driven and water-based migratory mechanisms in diverse microenvironments. Recent data reveal that the degree of cell confinement and the hydraulic resistance experienced by cells represent key factors in determining the mechanisms driving cell movement. Theoretical modeling utilizing a two-phase model of the cell cytoplasm also predicts that the hydraulic resistance experienced by the cell dictates the relative contribution of water flow/OEM to the observed cell speed. Mounting experimental evidence also suggests that cells can sense hydraulic pressure and modulate cell migration mechanisms. In this grant application, we propose to develop an integrated modeling and experimental approach to delineate the relative contributions of the actin-phase and the water-phase to cell migration as a function of external hydraulic resistance. In Aim 1, we propose to directly quantify how hydraulic resistance influences cell migration speeds by examining cells both in 2D in media with added methylcellulose, which increases medium viscosity, and inside confining microchannels of varying channel length, which also modulate hydraulic resistance. The roles of key ion channels and transporters that are involved in setting up water flux and the energetics of migration will be explored experimentally and theoretically. We will also identify the key mechanosensitive ion channels responsible for sensing hydraulic resistance. In Aim 2, we will explore the interplay between actin polymerization, membrane tension changes and OEM in environments of elevated hydraulic resistance. We will also extend the two-phase theoretical model of cell migration in include membrane tension and flows. Since cell migration speeds may depend on cell shape, in Aim 3, we will develop a general two-phase moving boundary method to compute cell movement for arbitrary cell shapes. We will also explore how OEM influences cell migration in dense vs more porous 3D collagen matrices, which exhibit different hydraulic resistances. Taken together, we will discover the mechanisms behind the counterintuitive observation of faster migration in high hydraulic resistance environments using a multidisciplinary approach, involving state-of-the-art microdevices, imaging, molecular biology tools along with mathematical modeling.